Regulation of gene transcription is key to understanding mechanisms underlying heart development, congenital heart disease, and developing therapies for heart regeneration post injury. Epigenetic modifications are key modulators of the transcriptional landscape, and as such, enzymes which catalyze these modifications have become a subject of great interest. In particular, histone modifications are thought to play key roles in multiple aspects of gene regulation, including mediating interactions with chromatin remodeling factors, docking of general or specific tissue specific transcription factors or cofactors, and perhaps mediating splicing. Histone acetylation and deacteylation have been intensely studied. More recently, histone methylation has come under scrutiny, and greater understanding of the role of specific methylation marks has emerged. The histone methylation mark histone H3 lysine 79 (H3K79), is uniquely catalyzed by the lysine methyltransferase, DOT1L. Multiple lines of evidence suggest critical and specific roles for DOT1L during cardiogenesis and in postnatal heart. DOT1L is highly expressed in the heart throughout development, and global ablation of Dot1L results in mid-gestation lethality with multiple cardiovascular abnormalities. Ablation of Dot1L in cardiomyocytes at mid-gestation with aMHC-cre results in cardiomyopathy in adult heart, accounted for in part by perturbation of dystrophin gene expression. Evidence from cardiomyocyte differentiation of mouse embryonic stem cells indicated that DOT1L plays a critical role at earliest stages of cardiomyocyte differentiation, yet no in vivo studies have addressed this issue. Accordingly, in preliminary studies, we have ablated Dot1L at E7.5 utilizing xMLCcre, and found that mutants exhibited cardiomyocyte hyperplasia, dying in the early postnatal period. Preliminary studies ablating Dot1L in perinatal mouse cardiomyocyte cultures demonstrated an ongoing requirement for Dot1L to regulate cardiomyocyte cell cycle. Preliminary studies have demonstrated enrichment of H3K79 methyl marks at cardiac gene loci in neonatal and adult myocytes. Together, these observations have led to our hypothesis that Dot1L is required in cardiomyocytes at embryonic, neonatal and adult stages for cardiomyocyte cell cycle regulation and progressive differentiation of cardiomyocytes. To test this hypothesis, our Specific Aims are: 1) To characterize the cardiac phenotype of early cardiomyocyte-specific Dot1L mutants. 2) To identify genes directly regulated, positively or negatively, by DOT1L mediated-H3K79 methylation in vivo. 3) To examine the requirement for Dot1L in postnatal and adult heart. Results of these studies will give mechanistic insight into the role of DOT1L in cardiomyocytes throughout distinct developmental stages and in postnatal and adult heart. Importantly, these studies promise to give insights into mechanisms by which epigenetic modifications regulate cardiomyocyte cell cycle, and are therefore likely to suggest therapeutic approaches for cardiac repair in both developing and adult heart. DOT1L inhibitors are currently being tested for treatment of MLL-associated leukemias, therefore our studies will be informative as to potential cardiac side effects of these inhibitors. Our studies will also address the future potential of DOT1L inhibitors for cardiac regenerative therapies.